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A simple 2D difference experiment is described that allows quantitative measurement of C-13-C-13 J couplings between backbone carbonyl and side-chain carbons. Precise 3J(CC) values were measured from data recorded in just 2 h for a 1-mM solution of the 20-kD complex between the protein calmodulin and a 26-residue synthetic peptide. The J couplings aid in determining the chi1 angles of valine, isoleucine and threonine residues, and in making stereospecific assignments of the Val C(gamma) methyl groups. Error analysis indicates that the uncertainty in the derived J couplings is generally less than ca. 0.3 Hz.
